Polycythemia vera (PV), idiopathic myelofibrosis (IMF), chronic myelogenous leukemia (CML) and essential thrombocythemia (ET) are classified as the chronic myeloproliferative disorders because their pathophysiology involves the clonal expansion of a multipotent hematopoietic progenitor cell with the over-production of one or more of the formed elements of the blood (1,2,3,4). However, with the exception of CML, these disorders lack a clonal marker, their pathogenesis is unknown, and their diagnosis therefore is dependent upon clinical criteria. Remarkably, in spite of their origin from a transformed clone, the mature circulating blood cells in these disorders are morphologically normal and, in contrast to CML, progression to acute leukemia is far less common in PV, ET and IMF. Furthermore, while these latter disorders can mimic each other clinically, they have distinctly different clinical courses and differ with respect to their treatment. Therefore, the identification of a diagnostic marker would be very useful.
Polycythemia vera is the commonest of the chronic myeloproliferative disorders and although its hallmark is trilineage hyperplasia, erythrocytosis is its most prominent clinical manifestation. For this reason, most investigators have focused on erythropoiesis in PV in an attempt to define its etiology but without notable success. There is a need in the art for diagnostic methods for distinguishing PV from other diseases involving erythrocytosis.
It is an object of the invention to provide methods of distinguishing polycythemia vera and idiopathic myelofibrosis from other diseases causing erythrocytosis.
It is another object of the invention to provide a method to aid in the diagnosis of polycythemia vera.
It is yet another object of the invention to provide an antibody useful in the diagnosis of polycythemia vera.
These and other objects of the invention are provided by one or more of the embodiments; described below.
In one embodiment a method is provided for distinguishing polycythemia vera and idiopathic myelofibrosis from other diseases causing erythrocytosis. The method comprises the steps of: contacting platelets or megakaryocytes of a patient with thrombopoietin; and determining whether the step of contacting with thrombopoietin causes proteins of the platelets or megakaryocytes to become phosphorylated on tyrosine residues; wherein a patient is identified as having polycythemia vera or idiopathic myelofibrosis when the step of contacting with thrombopoietin does not cause phosphorylation on tyrosine residues of platelet proteins, or wherein a patient is identified as not having polycythemia vera or idiopathic myelofibrosis when the step of contacting with thrombopoietin does cause phosphorylation on tyrosine residues of platelet proteins.
In another embodiment a method is provided to aid in the diagnosis of polycythemia vera. The method comprises the steps of: measuring thrombopoietin level of a blood sample isolated from a patient suspected of having polycythemia vera or idiopathic myelofibrosis; and comparing the measured level of thrombopoietin to the levels of thrombopoietin of a population of normal human controls; wherein a patient is identified as potentially having polycythemia vera or idiopathic myelofibrosis if the measured thrombopoietin level is more than two standard deviations above the mean value of the population.
In still another embodiment of the invention another method is provided for distinguishing polycythemia vera and idiopathic myelofibrosis from other diseases causing erythrocytosis. The method comprises the step of testing the ability of Mpl of platelets or megakaryocytes isolated from a patient suspected of having erythrocytosis to bind to thrombopoietin, wherein an Mpl which has diminished ability to bind thrombopoietin compared to wild-type Mpl indicates that the patient has polycythemia vera or idiopathic myelofibrosis, and wherein an undiminished ability to bind thrombopoietin compared to wild-type Mpl indicates that the patient does not have polycythemia vera or idiopathic myelofibrosis.
In yet another embodiment of the invention a method is provided for distinguishing polycythemia vera and idiopathic myelofibrosis from other diseases causing erythrocytosis. The method comprises the step of: determining the presence or absence of a wild-type Mpl in platelets or megakaryocytes isolated from a patient by subjecting proteins isolated from the platelets or megakaryocytes to two-dimensional gel electrophoresis, wherein an altered isoelectric focusing point of Mpl indicates that the patient has polycythemia vera or idiopathic myelofibrosis.
In another embodiment of the invention another method of distinguishing polycythemia vera and idiopathic myelofibrosis from other diseases causing erythrocytosis is provided. The method comprises the step of: determining the presence or absence of a wild-type Mpl in platelets or megakaryocytes isolated from a patient by subjecting proteins isolated from the platelets or megakaryocytes to isoelectric focusing gel electrophoresis, wherein an altered isoelectric focusing point of Mpl indicates that the patient has polycythemia vera or idiopathic myelofibrosis.
In yet another embodiment of the invention a method of distinguishing polycythemia vera and idiopathic myelofibrosis from other diseases causing erythrocytosis is provided. The method comprises the steps of: contacting platelets of a patient with thrombopoietin; and determining whether the step of contacting with thrombopoietin potentiates aggregation of the platelets; wherein a patient is identified as having polycythemia vera or idiopathic myelofibrosis when the step of contacting with thrombopoietin does not potentiate aggregation of platelets, or wherein a patient is identified as not having polycythemia vera or idiopathic myelofibrosis when the step of contacting with thrombopoietin does potentiate aggregation of platelets.
In still another embodiment of the invention an antibody preparation is provided. The antibody preparation specifically binds to the cytoplasmic domain of Mpl protein and does not specifically bind to the extracellular domain of Mpl.
In another embodiment of the invention a method is provided for distinguishing polycythemia vera and idiopathic myelofibrosis from other diseases causing erythrocytosis. The method comprises the steps of: determining in platelets or megakaryocytes isolated from a patient, subcellular localization of Mpl, a receptor for thrombopoietin, wherein a membrane bound form of Mpl indicates that the patient does not have polycythemia vera or idiopathic myelofibrosis, and wherein a cytosolic form of Mpl indicates that the patient has polycythemia vera or idiopathic myelofibrosis.
According to still another embodiment of the invention a method of distinguishing polycythemia vera and idiopathic myelofibrosis from other diseases causing erythrocytosis is provided. The method comprises the steps of: isolating nucleic acids from hematopoietic cells of a patient; and determining whether the nucleic acids encode a wild-type or mutant extracellular domain of Mpl, a thrombopoietin receptor, wherein a mutant extracellular domain of Mpl indicates that the patient has polycythemia vera or idiopathic myelofibrosis, and wherein a wild-type extracellular domain of Mpl indicates that the patient does not have polycythemia vera or idiopathic myelofibrosis.
In another aspect of the invention a method is provided for distinguishing polycythemia vera and idiopathic myelofibrosis from other diseases causing erythrocytosis. The method comprises the step of: utilizing an antibody which binds to the wild-type extracellular domain of Mpl, a thrombopoietin receptor, to determine the presence or absence of the wild-type extracellular domain of Mpl in platelets or megakaryocytes isolated from a patient suspected of having erythrocytosis, wherein the presence of the wild-type extracellular domain indicates that the patient does not have polycythemia vera or idiopathic myelofibrosis, and the absence of the wild-type extracellular domain indicates that the patient does have polycythemia vera or idiopathic myelofibrosis.
These and other embodiments of the invention provide the art with reagents and methods for specifically and simply distinguishing polycythemia vera from other diseases associated with erythrocytosis.